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We work with numerous educational institutions. Like everyone else, they are finding creative ways to do more computing with less funding. Desktop virtualization and thin client computing is a trend we see increasing to meet the demands of lower-cost solutions for 21st Century learning opportunities. The goal of any desktop virtualization or thin computing project is to lower your per-seat cost. While there are may ways to accomplish this goal, one of the lowest cost ways is to leverage solutions from NComputing. [Read more…]
Citrix XenServer 5.6
We use Citrix XenServer in our Datacenter for hosting virtual instances. We have been using Citrix since version 5.0 and really feel it enhances the products and services we are able to offer our customers. I recently participated in a few XenServer webinars highlighting these new features:
- Increased Scalability
- Dynamic Memory Control
- Heterogeneous Pools
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- Workload Balancing with Power Management
Many of these features sparked my interest. The Dynamic Memory Control allows you to set memory ranges for the virtual machines so you can have more memory allocated then what the machine physically contains. The memory on each of the virtual machines will adjust on the fly without rebooting. [Read more…]

subset: commoncrawl


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Prom tickets sold at all lunches
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Prom is less than a month away and ticket sales are even closer. No tickets will be sold at the door, so make sure to buy tickets on the sale days, April 17 through the 21 during lunch. Each ticket will be $60. In order for a senior to buy a ticket, they must have paid all fines and turned in any elevator keys. This year's theme is "Night of 1000 Lights".
"It's like the last senior event you can really enjoy together before graduation, so I feel like every senior needs to go," senior advisor Ms.Kirby says.


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Last weekend I went and skipped town to visit Iris and JBurns. Now it's been a lil' while since we posted real blogs because shit got busy around here, had to promote the infamous Triple X Tycoon after all. Anyway let's recap for good measure. Hi, I'm Doop.
And I'm the whatever here at Joy-Toilet.com. JBurns is the resident asshole. Soup is Chinese but not really. Iris is the cheerleader but I think she looks better in short-shorts, I mean, instead of pleated skirts. I'm sayin, those legs tho'. Speaking of, there's this book with legs on the cover called Sleeping Naked is Green that happens to be the semi-point of this blog so let's get to it.
Last week it snowed, a good bit up here in New York but not too much. Just enough to justify laying around naked for days on end. I remembered the aformentioned book, written by Vanessa Farquharson while vegging out. It brought me back, days at the library reading random shit to pass the time. Then I wondered if it could be taken a step further.
If sleeping naked is green, assuming that you're one person, than what if it was two people? What about sex, is that green too? Yes it fucking is, if:
A) You squeeze in as much sex as you can in between showers.
B) Take showers with your other half.
C) Fuck in the shower, since water can serve as lube. This means you're using less artificial lube which means less plastic waste.
D) She can skip the bra since it may not be doing her any favors anyway. Earth likes that. Less to wash.
E) He can start using straight razors for that face (or his legs? We don't judge) since you'll get a closer shave which in turn means you'll need to shave less which hey, might get you laid and we're back to A. On the other hand just grow your hair out..which might get you laid too. Nature didn't come with a rule book on that anyway.
F) Go on nature dates instead of clubbing. You'll probably end up fucking in the woods which means less sheet washing. That means less excessive water being used and hey, that's eco-friendly.
Wow that was all about sex. It's a good thing too. I mean literally. Now the condom thing is a grey area so tread carefully, we ain't touching that. Or you, so don't get too excited. Or do get excited. Whatever you do just remember, when you fuck, fuck for forest.
- Doop


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As a therapist, I provide a safe, confidential and supportive environment in which growth and healing can occur. I view therapy as a collaborative process, where we identify your goals and promote the development of your strengths, resources, and solutions. It is a place to learn about yourself, how you relate to others, and the ways in which your thoughts, feelings or actions can inhibit your growth or help you meet your goals.
My style is compassionate, client-focused, and interactive, informed by psychodynamic and cognitive behavioral schools of thought. I will work with you to help you make real, positive changes in your feelings, your behavior and your relationships.
Please call or email Dr. Chase for a psychotherapy consultation today.


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Mariah Carey and Selenis Leyva killed the red carpet in sexy cut-out gowns. Find out which other stars made our best dressed list this week.
MORE: 20 Celebrity Wardrobe Mishaps!
1. Mariah Carey
Whoa, baby! Mariah Carey turned her sexy all the way up at 27th Annual GLAAD Media Awards. The singer wore a glamorous Mark Zunino haute couture gown with a plunging neckline.
2. La La Anthony
La La Anthony has been slaying the red carpet game lately. The star looked incredible in a ruffled black gown at the 69th Annual Cannes Film Festival.
3. Dascha Polanco
You better work, Dascha Polanco! The OITNB star showed off her curvetastic figure in a sleeveless white cut-out dress at People en Español's 50 Mas Bellos party.
4. Gina Rodriguez
Gina Rodriguez flaunted her kickass new haircut in an off-the-shoulder white dress for the CW Network Upfront Presentation in New York City.
5. Lais Ribeiro
Lais Ribeiro looked beyond fabulosa at the amfAR Gala in France. The Victoria's Secret angel donned a gold cleavage-bearing gown, showing off her statuesque beauty.
6. Selenis Leyva
Selenis Leyva looked FIERCE at People en Español's 50 Mas Bellos party. The dominicana-cubana rocked a burgundy cleavage-bearing dress that perfectly showed off her hourglass figure.


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17 Greenidge Street, Wishart3 Bed 2 Bath 3 Car 622 m²
Located in the Mansfield State High School Catchment and Mansfield Primary School Catchment, this lowset brick home on an elevated 607sqm is perfectly positioned within walking distance to the schools and provides plenty of versatility for the discerning buyer.
Whether you're first home buyers, a growing family, investors looking to increase their property portfolio or someone looking for a property with scope to add value, then you needn't look any further than 17 Greenidge Street Wishart.
Inside, the home features a near new kitchen with glass splash back, stainless steel appliances, dishwasher, pantry and plenty of additional cupboard space. There is an air-conditioned formal dining area which flows out to a covered patio, informal living area and main air-conditioned living area, all with stunning hardwood parquetry flooring.
There is a built-in main bedroom with air-conditioning and en-suited bathroom, brand new main bathroom with separate shower and bath, two more built-in bedrooms plus an extra study with through-access to the garage.
The garage has been converted to include a multi-purpose room, perfect as a teenage retreat or home office and secure parking for one vehicle.
Outside, the homes leafy outlook provides plenty of peace and privacy, separate grassed areas for the kids to play, water tank and parking for two additional vehicles on the driveway.
Situated just a short stroll to Wishart Shopping Village, cafes and restuarants, supermarkets, medical centre, Boorabbin Picnic Grounds, bikeways, Bulimba walking track and within close proximity to public transport and the Gateway Motorway. You will absolutely love this fantastic home.
Additional Features:
Council Rates - approximately $1,820 per year
Water Rates - approximately $1,300 per year


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Executive Diploma in Dairy Science and Technology
The Dairy industries have opened up new avenues for both economy and employment. Adoption of the latest & advanced technology in milk processing can provide better quality, nutrition and more employment opportunities.
Fermented products such as cheese, concentrated milk, butter and even ice cream were discovered by accident but their history has also been documented for many centuries. These products have become popular among all cross section of populations irrespective of age group, and economic conditions.
In order to enhance the knowledge in the area of dairy, FFSQ, IGMPI has designed this programme of Dairy Science and Technology. This is undertaken through theoretical knowledge and practical skill as both are essential to master the field of dairy science.
The aim of the course is to understand the processing of milk and its properties, how to prepare milk products, their quality testing and also storage sanitation and effluent treatment. The course provides individuals with key knowledge of packaging, labeling and food safety. This course requires applying knowledge of science, technology and engineering to the processing and production of milk.
The course has been designed for professionals, students aspiring to work in the field of dairy, all involved in implementing and maintaining product quality systems, quality control and quality assurance personnel, new product development personnel.
Programme Structure
Module 1: Properties of milk
Module 2: Processing and quality parameters of milk
Module 3: Milk products-I
Module 4: Milk products-II
Module 5: Milk powder processing and milk substitutes
Module 6: Storage sanitation and effluent treatment
Module 7: Dairy analysis
Module 8: Packaging and labeling
Module 9: Food safety
Module 10: Practicals – analysis of raw material and finished products
Module 11: Laboratory demonstration, practicals and instrument handling
All those who have completed their graduation or post Graduation/ PhD are eligible to enroll for the course. As this course is truly professional and industry oriented, employed individuals from any sector (production, processing, quality, trial, R&D etc.) can also seek benefits of the course that have interest in food industry.
Programme Duration
Minimum time in which a student can complete this diploma course is six months while a maximum of one year is allowed to complete the course.
All the efforts will be made in the form of 6 weeks internship to provide industrial exposure to the students through internship with Food Companies so that they can have good practical knowledge beyond the classroom experience
The registration dates for this bi-annual program run by the institute are updated timely on the webpage. The training sessions and webinars are strictly scheduled depending on the availability of speakers. Effective E-learning tools incorporated into the design of the webpage make the course lectures, videos and study material easily accessible. This gives huge window of self-regulated and self-paced performance to the participants.
Programme Fee
The mandatory course fee (one-time payment) for the distance cum e-learning mode is Rs. 20,000/- (700 USD for overseas students) and Rs.45,000/- for the part time mode (weekend classes). This covers for the course registration fees, tuition fees, practical lab work, course material fees etc. Apart from this, an examination fee of Rs 400/- (USD 15) per module needs to be paid later as per the examination notification of the Institute.
Examination & Certification
All the participants are obliged to timely submit completed assessment assignments (during the course, usually after every module) and appear for an online exam at the end of the course. After successful completion, the participants will be awarded Executive Diploma in Bakery and Confectionery by Faculty of Food Safety and Quality, IGMPI. For all the above mentioned modules elaborate course material, self-assessment assignments and project work details would be provided by the Institute from time to time. Details get updated on the webpage as well.
Placement Assistance & Corporate Relations
The Institute has partnered with many organizations for providing with placement assistance to its participants. The robust placement cell comprises of senior level Human Resources professionals and Talent Acquisition experts which maintains close links with business and industry. We are engaged in promoting the employability of our participants by maintaining good rapport and relation with HR cell and recruiting managers of leading Food and Agriculture companies across the globe.
The efforts of our placement cell also include helping with professional resume writing, interview skills & conducting mock interviews etc.
Future career prospects
Faculty of Food Safety and Quality, IGMPIs e/distance course is a professional course targeted to cater the food industry, as there is a demand for trained food professionals. The information, guidance, practical training and completion certificate will provide the participant with not one but many opportunities in the industry. This would come true in form of job roles and positions like that of auditors, quality assurance executives, quality control executives quality managers, position in NPD team, food consultants, food journalism and many more.
For further enquiries or Prospectus, write to or call us on:
firstname.lastname@example.com/+91 8587838177


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Ph.D. candidate, Environmental Science Graduate Program, The Ohio State University, 2014-
B.S. in Mechanical Engineering, Valparaiso University, 2014
Research Interests:
Water-Energy Nexus
CO2-Geothermal Bulk Energy Storage
Research Experience:
January to July 2014 – Intern at Valparaiso University College of Engineering
- Designed, manufactured, assembled, and tested a heat transfer apparatus to measure the convection coefficient of water in a stainless steal crucible heated from below
Summer 2013 – Intern at Valparaiso University College of Engineering
- Performed an economic analysis of a solar thermal electrolytic magnesium production facility.
- Quantified Fe +2, Fe +3, and total iron concentrations in sulfuric acid in an attempt to elucidate the Magnetite to Hydrogen and Hematite reaction mechanism.
2014 Ohio State University Graduate School Fellowship
2015 NSF Graduate Research Fellowship, Honorable Mention
2016 NSF Graduate Research Fellowship, Honorable Mention
2016 ARPAe Innovation Summit, one of 100 students nationwide selected to attend
2016 OSU Environmental Policy Initiative Summer Research Grant ($4,000)
Other Interests:
Running, Reading, Music, Movies, and Baseball


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Wow the new catalog is amazing! I love all the hand-drawn images. Now I must decide what I get. Pick a petal is my very favorite. I also like Zoofari and the Punch Box. I have two parties to go to so I will be getting both of these...plus a few retired that I am buying!
I bought Delight in Life NUM off the SCS Buy/sell/trade for a great price, as well as frolicking frogs, and the 4 square punch that retired. Can't wait to play with these.
Monday, July 2, 2007
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Welcome to Abbey's Bakehouse
Our original Abbey's is located on the shores of Lake Rosseau in Minett, Ontario. The bakery finds its home in a 19th Century church, rebuilt and repurposed from its original location of Digby, Nova Scotia. Our sister location is situated in the heart of Port Carling.
Offering a variety of fresh breads, breakfast delights and decadent treats, Abbey's fulfills sweet-tooth cravings for cottage classics like homemade butter tarts, cookies and fresh fruit pies. Along with delicious sandwiches, and coffee, Abbey's also serves up handcrafted gelato and soft serve ice cream.
We invite you to come by and try our decadent treats made by people that are passionate about home baking and summertime Muskoka traditions.
See you at Abbey's.
We're Hiring for Summer 2018!
Looking for an amazing job opportunity this summer? If you are interested in an opportunity with Abbey's Bakehouse please email firstname.lastname@example.com and include "Abbey's Bakehouse Opportunity" in the subject line of the email.


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The soap opera, "One Life To Live" was canceled by its network a number of years ago, but the fans of this ground breaking drama have been hoping its return to the screens will be faster than they could have imagined. The soap opera initially made a fast return to the screens with new episodes being broadcast via an Online streaming service that made this one of the first to make a return after cancellation. Despite the legal problems that have paused the continuation of "One Life To Live" on a streaming service the drama is hopes to return in the near future.
There are many popular individuals who have made their mark in the world of acting and got their start in the acting profession with roles in "One Life To Live". An actress who has been making a name for herself in the TV and movie industry is Crystal Hunt, who made her name appearing as Stacy Morasco in the daytime drama. Hunt had already found success as an actress on the rival soap, "Guiding Light" before making the move to "One Life To Live".
Over the course of her career the Crystal Hunt has moved from starring in a range of drama's as a child to appearing as an adult in "One Life To Live" and a number of other dramas. Hunt has moved on like many of her fellow soap stars from the drama to make their way from the New York based soap opera to Hollywood. Crystal Hunt has recently been seen in the role of Lauren in "Magic Mike XXL", which has been one of the most critically acclaimed comedy drama movies of recent times. Crystal also has many more credits on IMDb.
The end of "One Life To Live" not only marked the move to an Online streaming service in one of the first productions designed specifically for streaming, but also marked the end of the production of soap operas in New York. "One Life To Live" marked many changes in the production of television and also brought to a close a specific period of time in daytime television.


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Boilers: A Practical Reference (Industrial Combustion) By Kumar Rayaprolu
2012 | 649 Pages | ISBN: 1466500530 | PDF | 44 MB
Following the publication of the author's first book, Boilers for Power and Process by CRC Press in 2009, several requests were made for a reference with even quicker access to information. Boilers: A Practical Reference is the result of those requests, providing a user-friendly encyclopedic format with more than 500 entries and nearly the same number of supporting illustrations.
Written for practicing engineers and dealing with practical issues rather than theory, this reference focuses exclusively on water tube boilers found in process industries and power plants. It provides broad explanations for the following topics:
A range of boilers and main auxiliaries, as well as steam and gas turbines
Traditional firing techniques―grates, oil/gas, and modern systems
Industrial, utility, waste heat, MSW and bio-fuel-fired boilers, including supercritical boilers
The scientific fundamentals of combustion, heat transfer, fluid flow, and more
The basics of fuels, water, ash, high-temperature steels, structurals, refractory, insulation, and more
Additional engineering topics like boiler instruments, controls, welding, corrosion, and wear
Air pollution, its abatement techniques and their effect on the design of boilers and auxiliaries
Emerging technologies such as carbon capture, oxy-fuel combustion, and PFBC
This reference covers almost every topic needed by boiler engineers in process and power plants. An encyclopedia by design and a professional reference book by focus and size, this volume is strong on fundamentals and design aspects as well as practical content. The scope and easy-to-navigate presentation of the material plus the numerous illustrations make this a unique reference for busy design, project, operation, and consulting engineers.
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Short Course: Unearthing Pompeii session one
William McMahon Ball Theatre Old Arts (Building 149)Map
More Information
6.15pm-8.30pm, Tuesdays 26 June - 31 July
Join us as we journey through the perpetual unearthing of Pompeii and examine its history, buildings, public and private spheres of everyday life, recreation, entertainment and art.
Session One
From the Beginning: The History of Pompeii and the Eruption of Vesuvius
The eruption of Vesuvius in AD 79 has preserved not only buildings, including their wall paintings and mosaics, but also items of everyday life that disappeared long ago from most of the Roman cities excavated elsewhere. At Pompeii (and Herculaneum) objects such as combs, mirrors and glass vessels have been preserved. Even the Pompeiians themselves and their animals survive in a series of remarkable plaster casts. From casts and wall-paintings, we are able to reconstruct the precise appearance of houses and shops, the beds and couches they sat upon, what they ate and the dinner services they used. At Herculaneum, not only are wooden beams, shop shutters, garden trellises and ceilings preserved, but even the bread, nuts and eggs that were to be eaten that day. Such is the wealth of information uncovered in these buried cities that we know from election posters who was standing for office in a particular year, from inscriptions the name of the architect of the theatre at Pompeii, and from graffiti people's opinion of various gladiators.
About the course
Over six consecutive weeks, join us as we journey through the discovery of the perpetual unearthing of Pompeii with comparisons and references to Herculaneum and other ancient buried cities. This short course will closely examine the history, the buildings, public and private spheres of everyday life, recreation, entertainment and art.
Each session includes two one-hour evening lectures with Q&A and a break with light refreshments. Course handouts and further reading material will be available throughout the duration of the course. Certificates of participation will be offered upon completion of the course.
Individual session, 2 hours each: $65 / $55^
Series pass, 12 hours: $375 / $300^
^ University of Melbourne alumni, staff and students


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Arup's innovative design for the world-class 55,000-seat national stadium is a model for future sustainable stadium design. From the outset the design ambition was to create a sustainable, fully integrated sports, entertainment and lifestyle hub, accessible to all.
Arup was enlisted to lead the architectural and engineering design of the Singapore Sports Hub, conceived as part of the greater Marina Bay Masterplan to reposition Singapore as a great place to work, live and play. The appointment followed expertise demonstrated by the firm in creating international landmark stadiums like the Beijing National Stadium (Bird's Nest), the Allianz Arena and the City of Manchester Stadium.
Addressing the issue of spectator comfort in the tropics, Arup designed an ultra-thin dome roof structure with a movable roof that extends over the pitch. The clever design provides shade during events and an energy-efficient spectator cooling system that delivers cooled air to every seat in the stadium. To maximise the stadium's utility, an innovative moving tier design allows the venue to accommodate a wide sporting calendar of events from cricket to football and the annual National Day Parade.
Versatility is a key component of the stadium design. By integrating moving tiers, palletised turf modules and a fully closing roof, the stadium can accommodate a wide calendar of sporting events like football, athletics and cricket as well as other events such as the National Day Parade.
The focus has been to achieve this flexibility within the design without compromising on the proximity and quality of view for spectators at a football match. The stadium will be the first in the world to combine the sports of football, athletics and cricket in the same arena.
The National Stadium will be one of the largest in the world. Clive Lewis, design leader for the project says, "We designed the stadium as an elegant dome spanning 310m – that's close to four A380 airplanes parked wing to wing. Not only is it wide, the ultra-thin shell roof structure is also very efficient and uses minimum materials for construction."
The National Stadium is a model for tropical climate design. It features an innovative energy-efficient spectator cooling system that delivers cooled air to every seat in the stadium. These pockets of cooled air will reduce energy use significantly, compared to a more conventional overhead cooling system.
The Sports Hub is Arup's latest landmark project in Singapore following the successful completion of the necklace of attractions around Marina Bay which include the Singapore Flyer, the Helix Bridge and the Marina Bay Sands® integrated resort.
The Sports Hub will reinforce Singapore's position as a major international sports destination, following the success of events like the F1 and the Youth Olympic Games.
Arup welcomes the appointment of four new Principals in the Australasia Region
A perfect circle at the water's edge, the building's geometric shape symbolises unity, stability and rationality.
Arup was commissioned to undertake the Design Manual for Road and Bridges (DMRB) Stage 1, 2 and 3 Scheme Assessments as well as statutory procedures for the project.
A technically-challenging 7.8km upgrade to one of Wales' most important highways
Total design for new football stadium for ACF Fiorentina in Florence.
Arup developed a numerical model of aero-acoustic resonance for ENI's headquarters in Milan.
Stunning rectracting roof at Singapore Sports Hub in action.
Singapore Sports Hub shines in the night.
Final demolition stages of Singapore's 37-year-old national stadium.


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Shreveport Regional Airport
The Shreveport Regional Airport has had a great start in 2013, with more travelers passing through the airport & traveling to hundreds of destinations around the world.
Last year, SHV began direct flights to Denver on United Airlines, opening a gateway to the West for the Ark-La-Tex region, in addition to their existing non-stop service to Houston. Shreveport Regional Airport also offers year-round non-stop flights to Las Vegas & seasonal service to Orlando on Allegiant, with low airfares attracting travelers from as far away as Dallas.
Along with our other air carriers, Delta and American Airlines offer flights to Atlanta, Memphis & Dallas, with connections to more than 130 destinations around the world.
Shreveport Regional Airport now features jet service on every flight every day, and in the future you can expect to see larger planes at the airport, making trips more comfortable.
Your experience at Shreveport Regional Airport is also improving with our new restaurant, Tailwind, which offers fresh sandwiches and hot food on both the airport's concourses and in the pre-security atrium location.
Check out our web site, www.FlyShreveport.com, where you can find daily fare sales to help you better plan your next trip out of Shreveport. You can also follow us on Facebook and Twitter!


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The militant national construction union faces fines of close to $1 million for disrupting work sites in NSW and Queensland, and allegedly inciting workers to wear shorts, defying safety rules.
The penalty judgments come in the wake of the Fair Work Commission's approval of a merger between the Construction, Forestry, Mining and Energy Union and the Maritime Union of Australia, with a total 144,000 members, combined revenue of $146 million a year and $310 million in assets.
The CFMEU and 19 officials were fined $817,500 after they shut down two major Brisbane worksites as part of a campaign allegedly aimed at forcing a company to sign the union's enterprise agreement.
The union and one of its officials were also fined $58,500 after allegedly inciting workers to wear shorts on a building site in Newcastle.
The Australian Building and Construction Commission (ABCC) took legal action against the CFMEU after it encouraged workers to wear shorts and short-sleeved shirts in defiance of construction company John Holland's safety policy, which required long sleeves and long pants to be worn.
The ABCC alleged CFMEU official Pomare Auimatagi encouraged workers at a construction site at the University of Newcastle to wear shorts and a short-sleeved shirt to work, in breach of the company's work, health and safety policy for The Prevention and Management of Heat Stress.
The Federal Circuit Court found the union had breached the Fair Work Act in January 2014 by defying the so-called "two longs" safety policy.
The ABCC alleged Mr Auimatagi told workers words to the effect that "others will tell you that you're using long sleeves for UV protection but you should have the choice.…I'm suggesting that you come to work and make a stand". It was also alleged he warned the project manager that if any worker was removed from the site then the whole site would stop work.
The Federal Circuit Court heard that a number of workers allegedly breached the safety policy by attending work in shorts and short-sleeved shirts.
After the project manager ordered a worker not to return to the site until he was wearing appropriate safety clothing, up to 50 workers allegedly stopped work for the rest of day.
In a penalty judgement on Thursday, Mr Auimatagi was fined $7,500 and the CFMEU was ordered to pay the maximum penalty of $51,000 "for deterrence".
Federal Circuit Court Justice Sylvia Emmett said the official's conduct had been found to be "nothing short of unconscionable" and the CFMEU's conduct to coerce John Holland into not exercising its workplace right to enforce its "two longs" safety policy was "reprehensible".
She said the union had an "appalling record of non-compliance and repeated contraventions of civil penalty provisions of industrial relations legislation".
ABCC Commissioner Stephen McBurney said the CFMEU's conduct had put the health and safety of workers at risk.
"The company's safety policy is one most Australians are familiar with. The unlawful conduct of the CFMEU official was undertaken in defiance of that policy," Mr McBurney said.
"I am concerned about a number of aspects of this case; firstly, the actions taken to undermine a policy designed to protect workers on site, secondly the CFMEU's failure to acknowledge the consequences of those actions, thirdly the lack of remorse or contrition, and finally the history of offending referred to in the judgment of the court."
In the Queensland case, Federal Court Justice Darryl Rangiah described the union's conduct as "deliberate, flagrant and systemic" with no attempts to comply with workplace laws.
'Scabs' and 'dogs'
Work stoppages took place at the $777 million Ennoggera Army Barracks and the $60 million QUT Kelvin Grove Campus two construction sites over several months in 2013.
The court heard that union officials tried to stop workers from entering the site and waved signs at them which said "gutless grubs", "weak as p-ss", "scabs" and "dogs". CFMEU Assistant State Secretary Jad Ingham allegedly said words to the effect that: "the sooner you sign the agreement, the sooner it will stop".
Mr McBurney said the level of intimidation directed at both the head contractor and the workers was "alarming".
"This was a protracted campaign orchestrated by repeat offenders intended to force the company to sign a CFMEU enterprise agreement," he said. "The penalties issued today by the Federal Court send a strong message that coercive and intimidatory behaviour will not be tolerated."
'Holding businesses ransom'
Workplace Minister Craig Laundy said that "unfortunately these matters occurred before legislation to reinstate the ABCC came into effect, which could have seen these penalties tripled".
"In recent years the courts have imposed fines of more than $13 million on CFMEU-related unions and officials for proven breaches of industrial law. As at 31 December 2017, there were 77 CFMEU officials before the courts," Mr Laundy said.
"We simply cannot afford this blatant lawlessness of the CFMEU, holding our businesses and economy to ransom."
A CFMEU spokeswoman said the union "is considering the decisions".
Anna Patty is Workplace Editor for The Sydney Morning Herald. She is a former Education Editor, State Political Reporter and Health Reporter. Her reports on inequity in schools funding led to the Gonski reforms and won her national awards. Her coverage of health exposed unnecessary patient deaths at Campbelltown Hospital and led to judicial and parliamentary inquiries. At The Times of London, she exposed flaws in international medical trials.
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A relationship banned under traditional law.
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Doji Innocents were those courtiers who fully embraced the Bushido, rejecting to lie and deceive even in court. [1] They focused on upholding Honesty even in the world of politics, avoiding falsehood at all costs and calling on the strength of their honor to lend their words weight. [2]
History Edit
The Innocents appeared in the early centuries, with little influence. While many among the Crane belived Bushido was a code of nearly impossible standards, others felt the clan's origins in honor and courage could not be compromised with dishonor, even for a greater good. [3]
Dojo Edit
The Gozoku era was a time when the clan's willingness to compromise its principles had pushed it to the very brink of destruction. The Innocents rebuilt the clan's reputation and political power during the reign of Hantei VII and Hantei VIII, and eventually they were finally permitted to establish their own dojo within the Doji Academy at Shizuka Toshi. [4]
Known Technique Edit
|This Crane Clan related article is a stub. That means that it has been started, but is incomplete. You can help by adding to the information here.


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Olshan Properties Appoints Vincent Bucci as Managing Director of Hilton Marco Island Beach Resort & Spa
February 24, 2017 1:57pm
Marco Island, FL (February 24, 2017) – Hilton Marco Island Beach Resort & Spa announced today the appointment of Vincent Bucci, as its new Managing Director. With an award-winning track record and an extensive background in hotel and food & beverage operations, he will be responsible for ensuring the successful re-launch of the storied 30-year-old resort as it finalizes a $40 million-dollar transformation re-defining island style with modern elegantly designed accommodations, a redesigned pool, and a brand-new dining experience, The Deck at 560 Bar & Restaurant.
"Vincent's reputation for outstanding leadership and effective management proceeds him, and we know he will bring a new level of luxury service to the Hilton Marco Island Beach Resort & Spa," said Andrea Olshan, CEO of Olshan Properties.
Bucci offers the team at Hilton Marco Island Beach Resort & Spa more than 37 years of hotel industry experience in the country's top markets. His roots began at the Adams Mark Hotel in Philadelphia as the food & beverage controller. He was quickly escalated to prestigious managerial positions at The Mayflower Hotel in Washington, D.C. and Morton's of Chicago in Columbus, Ohio and New York City. Serving in both area and property general manager positions, he held task force positions, developed training programs, and successfully led the transition of multiple hotels including Hyatt Regency Atlanta Perimeter, Hyatt Palm Springs and Doubletree Suites Nashville.
Prior to accepting his role with Hilton Marco Island Beach Resort & Spa, Bucci served as General Manager of the Hyatt Regency Westlake Plaza, where he led operations for the resort-like hotel.
Bucci holds an accounting degree from Peirce College in Philadelphia, and currently resides in Naples, FL with his wife Susan. He is a member of the National Restaurant Association, Certified Food and Beverage Executive – AHLA and he enjoys golf, sports and boating.
"I'm truly appreciative of the opportunity to oversee this beloved property," said Vincent Bucci, Managing Director of Hilton Marco Island Beach Resort & Spa. "From the warm-hearted staff to the re-defined modern facility, the resort truly epitomizes paradise on the Gulf."
Hilton Marco Island Beach Resort & Spa looks forward to introducing its guests to the leadership of Vincent Bucci. For more information, please visit www.hiltonmarcoisland.com.
Tags: hilton marco island beach resort & spa,
vincent bucci,
olshan properties
Hilton Marco Island Beach Resort & Spa is a full-service destination resort located on 10.5 beachfront acres of the world-class, white sandy beaches of Marco Island overlooking the Gulf of Mexico. Following a $40 million transformation, the storied 30-year-old resort continues to embody boutique South Florida style and create an intimate and superior vacation experience. Hilton Marco Island Beach Resort & Spa has state-of-the-art amenities and luxurious accommodations delivering consistent guest satisfaction since 1985. Recognized for its impeccable reputation in South Florida, the property is one of the most luxurious, award-winning and exclusive resorts, boasting several prestigious awards such as the TripAdvisor Certificate of Excellence 2015, AAA Four Diamond Award and Meetings and Conventions Gold Key Award 2015. To learn more, please visit www.hiltonmarcoisland.com and check out Facebook.
Olshan Properties is a privately owned real estate firm specialized in the development, acquisition, leasing and management of commercial real estate for over 55 years. The close integration of investment and operating capabilities has given Olshan Properties a reputation as one of the leading private owner/operators of commercial real estate in the country.
Olshan Properties currently owns and/or manages, individually or with its affiliated companies, a diverse portfolio of commercial properties including Retail, Hotel, Office, and Residential in eleven states. For more information, click here.
Contact: Anna Feiner
email@example.com /
Olshan Properties Appoints Steven Falciani as General Manager of Hilton Marco Island Beach Resort & Spa
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Hilton Marco Island Beach Resort & Spa Appoints New Executive Chef and Food & Beverage Director
Olshan Properties Set for January Debut of Hilton Marco Island Beach Resort Following $40M Renovation
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Olshan Properties Launching $40 Million Transformation of the Hilton Marco Island Beach Resort & Spa
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Olshan Properties Appoints Matthew B. Winn As Chief Operating Officer
Olshan Properties Promotes Alan Howe to VP & Carrie Richards to GM at Hilton Columbus at Easton, OH
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Underground presents Solomun... on Friday 9th June 2017
2 years ago we booked Solomun (Official) to play at our Bombed Out Church, this was probably one of the best events we have ever put on.
We have been working with Underground Liverpool to bring him back and we can now announce that Solomun will be playing an intimate gig at Underground on Friday 9th June.
Music Genres:House, Tech House, Techno
Please note: The event information above has been added by the organiser. Whilst we try to ensure all details are up-to-date we do not make any warranty or representation as to the accuracy or completeness of the information shown.
Underground presents Solomun... news
Solomun returns to Liverpool for an intimate party
20th Feb 2017 8:23pm
The Diynamic boss is heading to Underground in June - find out how you can ensare tickets.


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4 Bedroom House in Mtunzini
Dee Ayliffe
Lew Geffen Sotheby's International Realty-Zululand
4 Bedroom House in Mtunzini
Brand new spacious free flowing home on the Estate. Tastefully finished with top class fittings, modern and welcoming. Bedrooms are all en-suite . Triple garage with ample parking space.
The 175 hectare Zini River eco-estate is committed to conservation-orientated property development. It embraces and upholds the natural beauty of Mtunzinis local fauna and flora. Located on grassy reclaimed canelands, it is nestled into the indigenous forest that surrounds the Umlalazi River in Mtunzini. With beautiful sea, estuary and riverine forest views, Zini River Estate places focus on environmental rehabilitation and eco-conscious living, providing an excellent investment opportunity in the unspoilt nature of Zululand. The adjacent Umlalazi Nature reserve together with the planting of 10 000 trees, and a requirement for indigenous gardens, has created a truly natural home for wildlife and birdlife of over 300 species. Reedbuck and Impala have been introduced to the estate, and can be found wandering freely.
The burgeoning estate offer 263 freehold stands at roughly 1500sqm, 56 sectional title units and 13 large equestrian stands ranging from 7000 to 21000sqm. The careful positioning of individual stands has resulted in optimisation of river and sea views. A flexible architectural code making use of quality materials and subdued colours ensures the aesthetic appeal of the development whilst catering for individual taste. The development spans 85 hectares with the balance reserved for conservation. Over half the available land has been reserved for common use, to maximise the sense of space and freedom. River access, hiking and bike trails and an extensive programme for the rehabilitation of the indigenous vegetation, brings nature to your doorstep. The estate is dog friendly, however when walking they must be kept on leash, due to the wildlife.
Iliza Bay, the sectional title development within the estate, overlooking the sea and the Umlalazi estuary, offers unsurpassed lifestyle in a pristine setting. The design of the units creates a spacious flow between inside and outside living areas, making the most of the warm climate. It has proven to be an extremely popular investment opportunity for rentals in a secure lock up and go environment. Facilities in Iliza Bay include a swimming pool and tennis court as well as a clubhouse which can be hired for functions.
The estate offers bird watching, walking, fishing, canoeing, mountain biking and picnicking. The estate perimeter is fenced with monitored electric fencing with 24 hour security for total piece of mind. The estate has its own independent swimming pool and clubhouse for those not in Iliza Bay.
As the pearl in the Zululand crown, Mtunzini is close enough to big centres and yet far enough away to escape the rat race. Only 43km from Richards Bay, 87km from Ballito and 105km from King Shaka International Airport. It enjoys a sub-tropical malaria-free climate.
Mtunzini offers sporting enthusiasts a wide range of clubs and activities. These include the Mtunzini Athletic Club which offers road running and trail running the Mtunzini Country Club with its nine-hole golf course, tennis courts, squash court, fully-fledged gym, and a mountain biking section the Mtunzini Bowling Club and the Mtunzini Ski Boat Club. Hiking trails through the dune forest allow for peaceful reflections, and the Umlalazi lagoon is the perfect place for water skiing, canoeing, fishing and enjoying sundowners around a braai fire.
Nature lovers thrive on the wildlife and birdlife, of which many species are unique to this environment. Amongst others are the palm nut vulture, mangrove kingfisher, fish eagles, bushbuck, red duiker, fiddler crabs, mudskippers, vervet monkeys, and the dearly loved resident Zini zebbies.
Reputable pre-primary and primary schools and the availability of transport to excellent nearby high schools makes for excellent and affordable education opportunities.
With no traffic lights, little traffic and no shopping malls, Mtunzini is nothing short of amazing.
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Post-Doc in Mathematical Demography and Ecology, University of Amsterdam
A Post-Doctoral position is available with Prof. Hal Caswell, in the Theoretical Ecology Research Group at the University of Amsterdam.
This project focuses on developing matrix models for cohorts and populations, and applying them to demographic and biodemographic data on a variety of taxa, including humans. Specific questions involve frailty and other sources of heterogeneity; mortality, longevity, and disparity; multistate models; models for disease dynamics and health; population projections; and the effects of stochastic environments.
For more details, or to apply, please go to: http://www.uva.nl/en/about-the-uva/working-at-the-uva/vacancies/item/14-258.html
For information, please contact Hal Caswell
Deadline 15 September 2014.


text: Anne Miller recently completed her Master's degree in Public Policy and Public Administration from Concordia University in Montreal after interning at, and writing her thesis on, the International Criminal Court. Anne has worked significantly in program evaluation as a consulting associate with Brenda J Simpson and Associates, and has a strong background in statistical analysis, social return on investment accounting and logic model creation. She has also worked in a variety of policy areas, including indigenous peoples policy (with Dr. Daniel Salee at Concordia University), development policy (with the UNDP in South Korea), social policy (with Aspen Family Services and Brenda J Simpson and Associates), and environment policy (with Dr. David Swann, MLA for Calgary Mountainview). Currently she is the Research Coordinator for the Centre for Criminology and Justice at Mount Royal University.


text: Paige Roxbury Crop New line of denim at Ladybird.... Paige. Pomodoro color is arriving next week, we could not be more excited - woo hooo. What color should we pick up next? I am torn between the sea glass or jenny. Thoughts? Newer:jane birkin - so freshOlder:SALE PostedJune 25, 2011 AuthorLadybird Fashion CategoriesUncategorized


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Treasure Hunters (Hardcover)
Quest for the City of Gold
Jimmy Patterson, 9780316349550, 384pp.
Publication Date: January 15, 2018
List Price: 14.99*
* Individual store prices may vary.
Gear up for an exciting new adventure with the thrill-seeking Kidds as they search for a missing city in South America made entirely of gold When Bick and Beck Kidd find a hidden trove of pirate treasure, it includes a map with clues to an even bigger score: the lost Incan city of Paititi. But treasure hunting is never easy--and when the map is stolen, the Kidds must rely on Storm's picture-perfect memory to navigate the dangerous Amazon jungle. Watch out for that nest of poisonous snakes To save the Amazon rainforest and stop a Peruvian tribe from losing their home, the Kidds must unlock the secrets to the missing map and find the fabled city of Paititi...before the bad guys find it first. The race is on
About the Author
James Patterson holds the Guinness World Record for the most #1 New York Times bestsellers, and his books have sold more than 350 million copies worldwide. A tireless champion of the power of books and reading, Patterson created a children's book imprint, JIMMY Patterson, whose mission is simple: "We want every kid who finishes a JIMMY Book to say, 'PLEASE GIVE ME ANOTHER BOOK.'" He has donated more than one million books to students and soldiers and funds over four hundred Teacher Education Scholarships at twenty-four colleges and universities. He has also donated millions to independent bookstores and school libraries. Patterson invests proceeds from the sales of JIMMY Patterson Books in pro-reading initiatives.


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''I would hope that we won't capsize, but if we do, you need to grab Freddy and swim out from underneath,' said my dad as we sat huddled in the Wayfarer.
I looked at my stepbrother, he was eight, I was sixteen. He didn't look as concerned about this as I was. I hoped that sisterly instinct would kick in should we be plunged into the Solent's chilly May water and that I'd focus more on saving his life than panicking about my own. I eyed up the Lymington to Yarmouth ferry as it trundled past in a mess of wash and prayed we'd never have to find out.'
Upcoming book, In Bed with the Atlantic….
My first book, In Bed with the Atlantic, is coming out this autumn – telling the story of how I, a non-sailor, found myself sailing around the Atlantic.
Far from grabbing the adventure with both hands, I was dogged by doubt, a belief that I wasn't a 'sailor', never would be and that I was in no way capable of such an undertaking. I believed the ocean was out to get me, that weather needed to be battled and that I would be forever ruled by the anxiety that plagued me. And yet, in the face of my uncertainty, I discovered more about myself and the world then I could possibly have imagined.
This book covers the adventure that I undertook together with my partner on our small, 40 year old boat. I hope that it will allow others to see that they don't have to be 'born adventurers' and that even some of the most isolated parts of this world are open to you – all you need to do is go.


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Republican presidential candidate Donald Trump has been known to be a little harsh when it comes to his opponents, but according to one CBS journalist, he isn't the most "vindictive" candidate in the race.
CBS's "Face The Nation" moderator John Dickerson recently appeared on the "Hugh Hewitt Show" to discuss the 2016 presidential election.
During the show, Hewitt asked Dickerson who would be more vindictive toward their political enemies between Trump and Hillary Clinton — and his answer was definitely not expected.
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CBS has been known to be friendly to liberal Democrats like Clinton, and rarely do you hear a negative comment, especially when compared to a Republican — especially one as hated by the liberal media as Trump.
But Dickerson surprised us all when asked about who would be more "vindictive" between the two party leaders.
TRENDING: Hate: CNN Commentator Explodes Over Trump Supporter's Minority Son
"Ohhh, man, I don't know, I think Clinton," Dickerson said.
The CBS moderator acknowledged that the GOP candidate has been "very tough" on those whom he does not like, but he pointed out that Trump often "gets over it" and is able to move on, while Clinton tends to hold a grudge.
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Trump has had more than a couple of feuds with political opponents, but in most cases, he has been able to smooth things over after the fact, and in some cases, become quite close with them.
Dr. Ben Carson and Gov. Chris Christie both had their moments of disagreement with the now-presumptive GOP nominee, but both of them have now joined with Trump in efforts to beat the Democrats in November. In fact, both Carson and Christie have spoken highly of the candidate after they dropped out of the race.
Meanwhile, the Clinton campaign still has leftover resentment toward President Barack Obama from the 2008 Democratic primary that didn't end well for Clinton.
Listen to Dickerson's comments here:
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H/T The Daily Caller
Are you surprised to hear a CBS moderator say that Clinton would be more vindictive than Trump? Like and share on Facebook and Twitter and let us know.
Who do you think would be more vindictive toward political opponents -- Trump or Clinton? Scroll down to comment below.


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Photography is a tool to spread massages set up the meaning; It's a great tool for commemoration of the moment. From my type of view photography is the way to cross everything: Limits, culture, prejudice or even conflicts. I believe that I use this wonderful gift to make good for people and preserve those moments for them. I hope you will enjoy watching my photography work, It is my huge lifetime factory.
Nitzan gur.
Love in the winter
They wait for the rain to stop and come out for show us all them love.
I am so happy that i been there with them in this magical moment of love.
This shot we taken at "Agamon Hula" in the north of Israel.
What a great day and lovely couple !
Agamon Hula is unique and magical experience, in nature without partitions where you can observe thousands of unique birds, animals and plants, in their natural habitat.
I took this photo in last year with a great couple.
When the sun came down he grabbed her into hes hands and it was a magical moment of love.
Perfect couple and so much love and happiness .
They jumped and laughed and it was beautiful moment.
I love to take photos with a real couples and real love and to share those happiness moments.
This photo is a symbol of the beginning .
We took this photo in 04:00 am in the morning, before the sun rise.
there was excitement in the air, and love was all around us.
We took this photo in the "kinneret-sea" which is the source of all water in Israel.


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The Lockheed EP-3 is the signals reconnaissance version of the P-3 Orion, operated by the United States Navy.
|Signals Intelligence (SIGINT)
|National origin
|United States
|Primary users
|United States Navy
Japan Maritime Self-Defense Force
|Developed from
|P-3 Orion
A total of 12 P-3C aircraft were converted to replace older versions of the aircraft, which had been converted in the late 1960s and early 1970s. The aircraft are known by the acronym ARIES, or "Airborne Reconnaissance Integrated Electronic System".
The squadrons that flew the EP-3E also flew the Lockheed EC-121 Warning Star from 1962 to 1974 and the Douglas EA-3B Skywarrior from 1960 to 1991. There are 11 EP-3Es in the Navy's inventory, the last of which was delivered in 1997.
General characteristics
The information contained on this page is unclassified, approved for public dissemination and is released under CC-BY-SA Licensing Agreement.
The USCG Utility Boat, or UTB, is a standard utility boat used by the United States Coast Guard for a variety of inshore missions. The 41 ft (12 m) UTB is designed... Read More
The 47-foot MLB is the standard lifeboat of the United States Coast Guard (USCG). The 47′ MLB is the successor to the 44′ MLB. The 47' MLB is designed to weather... Read More


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The Hill's Jonathan Easley reports that Breitbart's fact checking of fake news from establishment journalists has led to a series of corrections and retractions — and "the high-profile nature of the errors hurts the media's credibility at a time when the press is under more scrutiny than ever before."
From The Hill:
A string of high-profile corrections and retractions by major news organizations on stories about President Trump or his allies have fueled more allegations of bias in the mainstream press.
The relationship between Trump and the news media regularly hits new lows, with insults flying between the two camps and reporters warning that the president is inciting violence against journalists.
But Trump's allies on the right believe the president's claims that the mainstream media is "fake news" have been bolstered by recent missteps. They point to recent admissions from top news outlets — including CNN, the New York Times and the Associated Press — that bombshell stories were either overcooked or included incorrect details.
Adding insult to injury: Breitbart News, the pro-Trump media outlet that is scorned by many in the mainstream press, has been fact-checking their mainstream counterparts with some success.
Read the rest of the story here.


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The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2–Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1–Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled‐coil to form two connected sub‐complexes with Mad2. The Mad2 C‐terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular 'safety belts'. We show that Mad1 is a competitive inhibitor of the Mad2–Cdc20 complex, and propose that the Mad1–Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1–Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1–Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1–Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C‐terminal tail is required for ligand release from Mad2, and that the 'safety belt' mechanism may prolong the lifetime of Mad2–ligand complexes.
The spindle assembly checkpoint monitors sister chromatid bi‐orientation in mitosis and prevents loss of sister chromatid cohesion in the presence of a damaged spindle (Hardwick, 1998; Shah and Cleveland, 2000). Mad1, Mad2, Bub1, BubR1/Mad3 and Bub3 associate with kinetochores in prophase and prometaphase to monitor attachment of sister chromatids to the spindle (Chen et al., 1996, 1998; Li and Benezra, 1996; Taylor and McKeon, 1997; Taylor et al., 1998). These proteins are conserved from yeast to metazoans, suggesting that the signal transduction pathways originating at unattached kinetochores are functionally and mechanistically similar in all eukaryotes. The target of the spindle checkpoint is the anaphase‐promoting complex (APC), a multi‐subunit ubiquitin ligase, whose activity ultimately determines loss of sister chromatid cohesion at the metaphase–anaphase transition (Zachariae and Nasmyth, 1999). APC inhibition requires a direct interaction of Mad2 with Cdc20, a positive regulator of the APC (Li et al., 1997; Fang et al., 1998; Hwang et al., 1998; Kallio et al., 1998; Kim et al., 1998; Wassmann and Benezra, 1998). Cdc20 mutants impaired in Mad2 binding do not arrest in metaphase upon Mad2 or Mps1 over‐expression in fission and budding yeast, respectively (Hwang et al., 1998; Kim et al., 1998). The importance of Mad2 is underlined by the observation that its gene is essential in mammals, and that the disruption of one Mad2 allele causes enhanced rates of chromosome loss, impaired checkpoint function, and may promote tumorigenesis (Dobles et al., 2000; Michel et al., 2001).
Mad2 also interacts with Mad1, a 718‐residue coiled‐coil protein (Hardwick and Murray, 1995; Chen et al., 1998, 1999; Jin et al., 1998). The Cdc20 and Mad1 complexes with Mad2 are exclusive, and occur in the absence of other proteins in vitro (Sironi et al., 2001; Zhang and Lees, 2001; Luo et al., 2002). Precisely how these complexes relate to each other is unclear, but a functional link is indicated by the inability of Mad2 and Cdc20 to interact in budding yeast strains lacking Mad1 (Hwang et al., 1998; Fraschini et al., 2001), and by the observation that Mad1 depletion severely affects the spindle checkpoint in mammalian cells (Luo et al., 2002). Thus, although Mad2 can interact effectively with Cdc20 in the absence of other proteins in vitro, binding of Mad2 to Mad1 makes this interaction possible in vivo. Dynamic studies indicated that Mad2 cycles at the kinetochore with a rapid turnover (Howell et al., 2000, 2001). Conceivably, Cdc20 may associate with Mad2 at unattached kinetochores and depart to prevent loss of cohesion on already bi‐oriented sister chromatids. The Mad2 binding sites on Mad1 and Cdc20 have been mapped using genetic and biochemical approaches (Hwang et al., 1998; Jin et al., 1998; Kim et al., 1998; Luo et al., 2000; Zhang and Lees, 2001), and recently it was shown that these otherwise unrelated proteins share an ∼10‐residue Mad2 binding motif (Luo et al., 2002). This suggests that Mad2 is transferred from one ligand to the other via competition. Binding of Mad2 to peptides encompassing the Mad1 and Cdc20 binding sites promotes a similar dramatic conformational change in the Mad2 C‐terminal tail (Luo et al., 2000, 2002), raising the possibility that Mad1 may prepare the Mad2 binding site for Cdc20 entry, increasing the on‐rate for this interaction (Luo et al., 2002).
We determined the crystal structure of the tetrameric Mad1–Mad2 core complex and analysed its structural and biochemical properties to find out how this complex may favour the Mad2–Cdc20 interaction. We find that Mad1 is a competitive inhibitor of Cdc20, and that the Mad1–Mad2 complex is engineered to slow down the rate of formation of Mad2–Cdc20. The Mad2 C‐terminal tail locks in the Mad1 ligand, and release of Mad2 likely requires its unfolding. We show consistently that previous binding of Mad2 to Mad1 decreases the rate of the Mad2–Cdc20 interaction in vitro relative to unbound (apo) Mad2. The Mad1 binding sites are buried at the interface of the Mad1–Mad2 tetramer, and an unperturbed Mad1–Mad2 tetramer will not release Mad2. A 1:1 complex, however, will allow a slow exchange of Mad2 from Mad1 for Cdc20 binding. These results suggest that an appropriate conformational change in the Mad1–Mad2 complex may allow regulated release of Mad2 for Cdc20 binding, which our thermodynamic studies indicate as a favourable interaction.
Structure determination
A soluble complex between the C‐terminal region of human Mad1 (residues 485–718, Mad1485–718) and histidine‐tagged (His) human Mad2wt was purified from bacteria (Sironi et al., 2001). The complex eluted in a single peak from a size‐exclusion chromatography (SEC) column, but further analyses established that it contains a super‐stoichiometric amount of Mad2wt, probably due to Mad2 aggregation (Sironi et al., 2001). Consistently, Mad1485–718–Mad2wt was polydisperse (not shown) and did not crystallize. Mad2R133A, a point mutant of Mad2 with a substitution of Arg133 by alanine, effectively binds Mad1 and Cdc20 (Sironi et al., 2001). Its interaction with Mad1 is tight and results in a monodisperse complex (not shown) devoid of the excess Mad2 observed in the complex containing Mad2wt (Sironi et al., 2001). We refer to the resulting complex as the Mad1–Mad2 core complex. Although we were unable to crystallize Mad1485–718–Mad2R133A (large Mad1–Mad2 core complex), a complex containing a shorter fragment of Mad1 (Mad1485–584–Mad2R133A: short Mad1–Mad2 core complex) yielded a crystallizable sample. The structure was determined at 2.05 Å resolution using SIRAS methods on a selenomethionyl derivative (Table I) as described in Materials and methods.
Overall view of the Mad1–Mad2core complex
Mad1–Mad2core is a tetramer containing two copies of each subunit (Mad2A and Mad2B, yellow in Figure 1; and Mad1A and Mad1B). The Mad1 molecules are elongated, and are tightly held together by a parallel coiled‐coil involving the N‐terminal α1 helices (cyan). The Mad1 chains part in opposite directions at the end of the coiled coil, and point towards their Mad2 ligands, from which they emerge ∼20 residues later with another helical segment (α2, in green). The segment between the α1 and α2 helices of Mad1 (dark blue) contains the critical residues for Mad2 binding. The first part of this segment (the 'linker'; residues 531–539) is flexible and adopts different conformations in Mad1A and Mad1B, as revealed by superposition of the α1 helices (Figure 1C). As a result, the Mad1–Mad2 core complex is significantly asymmetric. The second part (residues 540–549) contains the Mad2 binding motif, which supports essentially identical interactions defining Mad1A–Mad2A and Mad1B–Mad2B sub‐complexes. The C‐terminal tails (residues 161–205) in both Mad2 monomers undergo a conformational change similar to that observed in the solution structure of Mad2 with the Mad2 binding peptide 1 (MBP1) (Luo et al., 2002).
The Paircoil server (Berger et al., 1995) suggests an uninterrupted coiled coil for the N‐terminal segment of Hs Mad1 not included in our structure (residues 1–484). There are essential segments in this region, which may be required to localize Mad1 at kinetochores (Chen et al., 1999). The prediction also suggests that after a short interruption around residue 584 (which was chosen to end our construct), a segment with a high propensity for coiled‐coil formation will resume until residue 630. The geometry of the α2 helices of Mad1A and Mad1B as they emerge from their cognate Mad2 ligands is incompatible with the formation of a parallel coiled coil. In the crystals, these helices pack with an anti‐parallel orientation with equivalent segments (α2′) in a second tetramer (Figure 1E). Although this interaction is weak (because Mad1485–584–Mad2 remains tetrameric in solution), it suggests that the α2 helices may form an intra‐molecular anti‐parallel coiled coil with a speculative α3 helix lying between residues 590 and 630, with a connecting loop tentatively fitted to residues 579–589.
To test this hypothesis, we introduced hydrophilic residues in place of the hydrophobic residues involved in the stabilization of the α2–α2′ interface observed in the crystals (Figure 1F). While the wild‐type (wt) sequence of Mad1485–718 was pulled down by His‐Mad2 from the bacterial lysates after co‐expression in bacteria (lane 2), the resulting Mad1485–718 mutants were unable to interact with Mad2 (lanes 3–5). The same mutations, however, were completely silent when introduced into Mad1485–584 (lanes 9–11). This striking difference could be explained if the mutated residues were exposed to solvent in Mad1485–584 and buried in Mad1485–718, because in this case hydrophobic to hydrophilic substitutions would be expected to cause structural destabilization in the latter but not in the former. These results, together with the structural prediction indicating the possible existence of an α3 segment, suggest that the α2 helices may indeed participate in an intra‐molecular α2–α3 anti‐parallel interaction similar to that observed in the crystals. A conserved RLK (Arg‐Leu‐Lys) motif in the putative α3 helix mediates binding of Bub1–Bub3 to Mad1–Mad2 in budding yeast (the corresponding motif in Hs Mad1 is located at residues 617–619), and mutational abrogation of this interaction prevents checkpoint activation without impairing Mad2 binding (Brady and Hardwick, 2000). The cartoon in Figure 1G shows that Mad1 may consist of a long parallel coiled coil leading into two head domains, each containing a Mad2 binding site followed by an anti‐parallel coiled coil, and by a C‐terminal ∼90‐residue region (grey) previously identified as a target of mutations abolishing the checkpoint (Chen et al., 1999). Resistance to limited proteolysis (not shown) suggests that the C‐terminal domain of Mad1 folds into a stable domain. We believe that the 2–2 tetrameric assembly revealed by our crystal structure is the functional Mad1–Mad2 species in vivo.
A 'safety belt' binding mechanism
The structure of the Mad1–Mad2 complex confirms the recent finding that Mad1 and Cdc20 share a common Mad2 binding motif (Luo et al., 2002). The details of the interaction of Mad2 with the two Mad1 motifs in the core complex are essentially identical, and binding promotes a remarkable reorganization of the Mad2 C‐terminal tail (residues 161–205; the red segment in Figure 2A) compared with the structure of apo‐Mad2 reported by Wagner and collaborators (Luo et al., 2000). The first 160 residues of Mad2 are structurally invariant in apo‐ or Mad1‐bound Mad2, but the C‐terminal region (starting from the β6–β7 loop, the 'hinge loop' in Figure 2B and C) occupies the opposite edges of the large exposed β‐sheet in these structures. In the complex, the Mad2 C‐terminal tail crosses the entire surface of the β‐sheet and locks Mad1 in such a way that it resembles a fastened safety belt. This is clarified by the surface representation in Figure 2C, showing how the Mad1 chain is 'threaded' through Mad2.
A structural alignment of the Mad2‐binding motif with the MBP1 peptide, whose solution structure in complex with Mad2 was recently reported (Luo et al., 2002), is shown in Figure 3A. We note a less conserved version of the motif in the APC activator CDH1, a target of Mad2B/Rev7 (Chen and Fang, 2001; Pfleger et al., 2001). Point mutations in the Mad2 binding motif abolish the Mad2–Cdc20 interaction and the spindle checkpoint in budding yeast (Hwang et al., 1998; Kim et al., 1998). To test whether analogous mutations in Mad1 affected Mad2 binding, residues Lys541, Leu543, Met545 or Pro549 were mutated into alanine, and the amount of wild‐type and mutant Mad1485–718 co‐purifying with immobilized His‐Mad2 was evaluated. Mutation of Lys541 (not shown) and Pro549 totally abolished binding, while the other two mutants displayed a partial but significant reduction in binding (Figure 3B).
A comparison of the Mad2 complexes with the MBP1 peptide (Luo et al., 2002) and with Mad1 identifies substantial similarities, but also important differences (Figure 3C). Besides forming a number of van der Waals and polar contacts with Mad2, both ligands bind by adding an (anti‐parallel) β‐strand at the end of the exposed β‐sheet of Mad1, a binding mechanism defined as β‐augmentation (Harrison, 1996). The MBP1 peptide, however, adopts a more extended and relaxed conformation relative to Mad1. The structural alignment shows that Ser1 (S1) and Pro8 (P8) of MBP1 occupy essentially identical positions to those of Lys541 (K541) and Pro549 (P549) of Mad1. Thus, nine Mad1 residues must be accommodated within the Mad2 binding site, rather than eight in MBP1. The Mad1 chain bulges out around residues 545–548, pushing against the Mad2 hinge loop (Figure 3C). As a result, the anti‐parallel pairing of the Mad1 chain against the β6 strand of Mad2 and against a segment of the Mad2 hinge loop is imperfect, and restricted to a smaller number of inter‐chain hydrogen bonds relative to the Mad2–MBP1 complex (Figure 3C). This might explain at least in part why Mad1 and Cdc20 synthetic peptides, which have a similar sequence and identical spacing between the lysine and proline residues (K541 and P549 in human Mad1), are significantly weaker Mad2 ligands than the MBP1 peptide (Luo et al., 2002). The fact that the phage display strategy used by Luo et al. (2002) identified sequences (such as MBP1's) that are distinct from those in Mad1 and Cdc20 may suggest that the interaction of Mad2 with the Mad1 and Cdc20 Mad2 binding motifs is designed to be sub‐optimal.
It should be noted, however, that the Mad1–Mad2 interaction is stabilized by a number of additional contacts in the tetramer, several of which involve the Mad1 linker region upstream of the conserved portion of the Mad2 binding motif. Because the linker regions are asymmetric, these contacts are different in the two sub‐complexes (Figure 4A and B). Mad1A residues 527–539 are involved in a number of additional interactions with Mad2A (Figure 4A, left), which are not observed in the Mad1B–Mad2B sub‐complex (Figure 4A, right). The Mad1B linker (residues 530–538), however, compensates for the fewer contacts by packing tightly against the Mad1A–Mad2A interface as shown in Figure 4B. Despite the asymmetric nature of the complex, whose origin remains unclear but which seems to rely on the flexibility of the linker, both ligand binding sites of Mad2A and Mad2B are deeply buried at the interface between the Mad1–Mad2 sub‐complexes in the tetramer (Figure 4C and D). As shown below, this organization significantly increases the stability of the Mad1–Mad2 complex. We predict that the release of Mad2 from the tetrameric complex implies opening of the structure to expose the Mad2 C‐terminal tails. This may occur along a preferential 'fracture' line determined by the asymmetry of the complex. Thus, although we do not demonstrate here that the asymmetry of the core complex is functionally important, we propend in favour of this hypothesis.
Insights into the Mad2 exchange reaction
The finding that Mad1 and Cdc20 share a conserved Mad2 binding motif argues that the Mad1‐dependent reaction that takes Mad2 on to Cdc20 occurs via a direct competitive mechanism. The structure suggests that Mad1 cannot be released without prior unlocking of the C‐terminal tail. Mad2 residues Val181, Leu183, Phe186, Ile190, Val193, Val197 and Tyr199 pack against the Mad2 hydrophobic core stabilizing the closed conformation. Two of these residues (Val197 and Tyr199), and another two (Met196 and Ala198) that are directly involved in the interaction with Mad1, fall within a C‐terminal 10‐residue segment whose deletion (Mad2ΔC) abolishes Mad1 and Cdc20 binding to Mad2 (Luo et al., 2000; Sironi et al., 2001). In apo‐Mad2, these residues are disordered in solution, and are not required for structural stabilization (Figure 2D; Luo et al., 2000). This likely explains why Mad2ΔC is structurally stable but impaired in ligand recognition (Luo et al., 2000; Sironi et al., 2001). As expected from the presence of a similar Mad2 binding site, the C‐terminal tail of Mad2 may adopt a similar conformation in the Mad1 and Cdc20 complexes (Luo et al., 2002). Unless tail opening occurred on a fast time scale, the transition from a Mad1–Mad2 to a Cdc20–Mad2 complex would be limited by the dynamics of tail opening, independently of the relative affinity of Mad2 for Cdc20 and Mad1. Because the two Mad1–Mad2 binding sites are buried, they may be inaccessible to Cdc20 within the tetrameric complex (Figure 4). These considerations raise the question of whether the postulated exchange reaction taking Mad2 from Mad1 to Cdc20 occurs spontaneously. To answer this, we designed a set of experiments to dissect the contribution to the binding mechanism of (i) the relative preference of Mad2 for the Mad1 and Cdc20 binding motifs, (ii) Mad1 locking by the Mad2 C‐terminal tail, and (iii) stabilization of the Mad1–Mad2 interaction as it might occur in the tetramer.
First, we determined thermodynamic parameters for the interaction of Mad2 with synthetic peptides corresponding to the isolated Mad2 binding motifs of Mad1 and Cdc20 using isothermal titration calorimetry (ITC; Table II). The Mad1 sequence used in these studies is unlikely to support self‐oligomerization, as it lacks the coiled‐coil region. It has also been shown that the interaction of Mad2 with Cdc20 and Mad1 peptides gives rise to simple 1:1 complexes (Sironi et al., 2001; Luo et al., 2002). Thus, our thermodynamic studies likely measure 1:1 interactions between Mad2 and its ligands. We refer to these interactions as 'monovalent' to distinguish them from those occurring in the Mad1–Mad2 tetramer, which are significantly tighter (see above and below). As shown in Table II, Mad2 binds the Cdc20 motif with 10‐fold higher affinity (KD = 100 nM) than the Mad1 motif (KD = 1.0 μM). We also assessed whether the R133A substitution in Mad2 had any effect on ligand binding. Table II shows that Mad2wt and the monomeric Mad2R133A bind their Mad1 and Cdc20 ligands with essentially identical affinity, confirming the non‐invasive nature of the R133A mutation. Although a structural explanation of why Mad2 binds the Cdc20 peptide more tightly than Mad1 is not available, the calorimetric determinations argue that Mad2 exchange to Cdc20 from a monovalent Mad1–Mad2 complex is a favoured reaction, provided no further stabilization of Mad1–Mad2 was present.
Preliminary unfolding of the Mad2 C‐terminus may be required for ligand release, and the closed conformation is expected to delay the disruption of the Mad2–ligand complex. To test this, we investigated whether the ability of a Cdc20 synthetic peptide to compete with a monovalent Mad1–Mad2 complex was sensitive to the initial state, locked or unlocked, of the C‐terminal tail. We considered apo‐Mad2 (Figure 2A) as representative of the unlocked state, while the locked state was represented by a Mad2 sample already bound to Mad1. A graphical representation of the experiment is shown on the right‐hand side of Figure 5A. GST–Mad1523–550 immobilized on a solid phase was mixed directly with recombinant apo‐Mad2R133A (Mad2 unlocked) in the presence of different concentrations of Cdc20 peptide, or pre‐incubated for 60 min with apo‐Mad2R133A to allow complex formation (Mad2 locked), and only then incubated with Cdc20. The competition reaction was protracted for either 15 or 300 min for both samples, at the end of which the bound proteins were isolated by centrifugation, washed and examined by SDS–PAGE (Figure 5, left panels). In the absence of pre‐incubation, the patterns were identical after 15 or 300 min (compare lanes 8–11 in the two gels), suggesting that the competition reactions reached equilibrium within the 15 min time frame. Pre‐incubation of GST–Mad1523–550 with Mad2, however, significantly decreased the ability of the Cdc20 peptide to compete for Mad1‐bound Mad2 within the 15 min time frame, and even after an incubation of 60 min we detected a significant difference in the pre‐ and non‐pre‐incubated samples (data not shown). These differences were completely eliminated after 300 min incubations (left bottom panel), indicating that this time is sufficient to equilibrate the competition reaction. Similar levels of Mad2 binding were observed in the pre‐incubated and non‐pre‐incubated samples in the absence of competing peptide (compare lanes 4 and 8) even after 15 min, showing that this time is sufficient for Mad2 to bind Mad1. Release of pre‐incubated Mad2 from Mad1 at 15 min was also dependent on the concentration of competing peptide, suggesting that besides tail opening, the release of the bound ligand is also limiting for the exchange reaction. Thus, the off‐rate of the Mad1–Mad2 interaction must be significantly slower than the on‐rate of the Cdc20–Mad2 interaction, even if this is a rather slow reaction per se (Sironi et al., 2001; Luo et al., 2002). The result also shows that Cdc20 and Mad1 are respective competitive inhibitors of their complexes with Mad2, and suggests that the conformational change of the C‐terminal tail is designed to slow down the release of a bound ligand.
The competition experiments were carried out with a monovalent Mad1 fragment. To establish whether the Mad1–Mad2 interaction was further stabilized in the tetramer, we asked whether the Cdc20 peptide was able to release Mad2 from Mad1–Mad2core. We incubated short Mad1–Mad2core (at a final concentration of ∼200 μM) with a 1 mM concentration of a synthetic peptide containing the Mad2 binding site of Cdc20, and after variable incubation times finally resolved the resulting species by size‐exclusion chromatography (SEC). In control experiments, the short Mad1–Mad2core, Mad2R133A and the Cdc20 peptide eluted as single peaks from a SEC column, as shown by their superimposed elution profiles in Figure 5B. Recovery of Cdc20 peptide in the Mad2 peak (Figure 5C, lanes 4–6) indicates that the Mad2–Cdc20 complex can be easily detected using this approach. Even after 16 h incubations, the Cdc20 peptide did not associate with Mad1–Mad2, nor did it disrupt this complex, as it might have been revealed by the appearance of Mad2–Cdc20 or free Mad1 (Figure 5D). Similar experiments with the long Mad1–Mad2core gave identical results, with no association or disruption of the complex upon incubation with Cdc20 (Sironi et al., 2001). Lack of exchange is unlikely to result from the use of a truncated form of Cdc20, because Cdc20 fragments containing the Mad2 binding site are tighter Mad2 ligands than full‐length Cdc20 (Tang et al., 2001; Zhang and Lees, 2001). This shows that the short Mad1–Mad2 core complex is very tight and indistinguishable from its larger counterpart, and confirms the structure‐based prediction that tetrameric Mad1–Mad2 is significantly more stable than a monovalent complex.
Mad1 interacts with Mad2 by β‐augmentation, a frequent binding mechanism in protein–protein complexes. The very atypical feature in Mad1–Mad2 is the refolding of the Mad2 C‐terminal tail over the Mad1 ligand in a way that is reminiscent of a fastened safety belt. We are unaware of a similar binding mechanism in other proteins. The Mad2–Cdc20 complex is expected to have an organization similar to that of Mad1–Mad2, with an elongated Cdc20 segment (residues 123–137) 'threaded' through the Mad2 C‐terminal tail.
Important conclusions from this study are (i) that monovalent Mad1 is a weak competitive inhibitor of the Mad2–Cdc20 complex, (ii) that the conformation of the Mad2 C‐terminal tail introduces a kinetic barrier towards the release of Mad2 from its ligand complexes, as a consequence of its required unfolding, and (iii) that the tetrameric assembly of the Mad1–Mad2 complex reinforces the interaction, preventing the release of Mad2. As expected for a competitive inhibitor, Mad1 opposes Mad2 binding to Cdc20 (Figure 5). Mad1 is essential to establish a Mad2–Cdc20 complex in vivo (Hwang et al., 1998; Fraschini et al., 2001), and the discovery that it acts as a competitive inhibitor of Mad2–Cdc20 is counterintuitive. Understanding why Cdc20 and Mad2 are unable to interact in vivo in the absence of Mad1, despite their proven record as direct ligands in vitro, is now a very urgent question. Mad2 binds poorly to full‐length Cdc20, suggesting that the binding site is shielded in the full‐length protein (Tang et al., 2001; Zhang and Lees, 2001). The activated Mad1–Mad2 complex may help Cdc20 to expose its Mad2 binding site. Furthermore, Cdc20 is associated with cellular chaperones, which may limit its ability to interact with Mad2 in vivo (Ho et al., 2002; Pines, 2002). Topological considerations may also be important to limit the Mad2–Cdc20 interaction. Mad1 recruits Mad2 to kinetochores, where the Mad2–Cdc20 interaction is expected to take place (Chen et al., 1998; Luo et al., 2002). It is unclear, however, why Cdc20, which is localized to kinetochores in a Mad1‐independent manner (Shah and Cleveland, 2000; Sironi et al., 2001; Luo et al., 2002), is ineffective in directly recruiting Mad2 to these structures. The concentrations of Mad2 and Cdc20 may be insufficient to drive complex formation, while Mad1 may be able to recruit Mad2 to kinetochores through a high‐affinity binding site. Subsequently, an external input of energy may destabilize the Mad1–Mad2 complex, allowing the release of Mad2 at a sufficiently high local concentration to support the interaction with Cdc20 (Figure 6). We suggest that the role of Mad1 as a matchmaker for the Mad2–Cdc20 complex in spite of its role as a competitive inhibitor of this interaction can be explained if funnelling through Mad1 is the only way in which Mad2 can effectively reach Cdc20 in vivo.
Previous studies have indicated that an unperturbed, stable Mad1–Mad2 complex exists during the cell cycle (Shah and Cleveland, 2000). As suggested above, this complex may be destabilized at kinetochores by the spindle checkpoint to promote the transfer of Mad2 to Cdc20, and the subsequent recruitment of new Mad2 from a cytoplasmic pool (Figure 6). Microtubule attachment may restore an inactive form of Mad1–Mad2, preventing further dispensing of Mad2 into APC inhibitory complexes, eventually dampening checkpoint signalling. Because the affinity of an isolated monovalent Mad1–Mad2 interaction is significantly lower than those occurring in the tetramer, the conversion of Mad1–Mad2core to a dissociating form may be promoted by a conformational change disrupting the interaction of the globular heads observed in the tetrameric assembly, and resulting in the exposure of the Mad2 ligand binding sites for ligand exchange. After Mad2 release, the regeneration of a high‐affinity binding site may recruit new Mad2 to restore the high‐affinity unperturbed Mad1–Mad2 core complex. The cycle may continue until the checkpoint is active, sustained by metabolic energy. The high turnover rate of Mad2 at kinetochores during checkpoint activation may reflect the kinetics of this reaction (Howell et al., 2000, 2001). Positive regulators are expected to play an essential role in the postulated exchange reaction. The suggestion that the Bub1–Bub3 complex binds Mad1–Mad2 near the Mad2 binding site lends further credit to this kinase, which phosphorylates Mad1 in vitro (Seeley et al., 1999), as a possible regulator of Mad1–Mad2. Other actors in the exchange reaction may be the BubR1–Bub3 complex, which binds Cdc20 and may enter a quaternary complex also containing Mad2 (Fraschini et al., 2001; Sudakin et al., 2001; Tang et al., 2001), and the Mps1 kinase, which phosphorylates Mad1 in budding yeast (Hardwick et al., 1996). Our work shows that the C‐terminal lock must open before a ligand can be released from Mad2. This observation rules out the proposal that Mad1 favours binding of Cdc20 by Mad2 by stabilizing a pre‐formed template with a reorganized C‐terminal tail (Luo et al., 2002). The 'safety belt' binding mechanism may be useful to delay the disruption of the Mad2–Cdc20 complex once this is formed at activated kinetochores. The mechanism may allow the generation at the kinetochore of a kinetically stable Mad2–Cdc20 complex, despite the low binding affinity required to prevent significant direct binding in the absence of an activated checkpoint.
An in vitro approach aiming at the mechanism by which Mad2 is routed from a soluble pool to Mad1 and then to Cdc20 will have to cope with the fact that simple mixing of detectable concentrations of Mad2 and Cdc20 in vitro will result in direct binding. The hypothesis that Mad2 is funnelled to Cdc20 via Mad1, however, presents several testable implications in vivo. The rate of Mad2 release from the Mad1–Mad2 complex must increase upon checkpoint activation, while a high binding on‐rate would be required to replenish Mad2 at the kinetochore upon checkpoint activation. It should be possible to identify mutations in Mad1, Mad2 or other spindle checkpoint proteins that do not affect the interaction of Mad2 with Mad1, but rather the release of Mad2 from this complex to bind Cdc20. A likely candidate for such an 'exchange mutant' in Mad1 has been described above (Brady and Hardwick, 2000). Furthermore, because Cdc20 acts as the ultimate receptor for Mad2, the dynamic behaviour of Mad2 at kinetochores should be affected by Cdc20 depletion. Measurements of Mad1, Mad2 and Cdc20 dynamics at different stages of the cell cycle and with appropriate mutational strategies may be used to test this hypothesis.
Materials and methods
Structure determination
Mad1–Mad2 was generated and purified essentially as described (Sironi et al., 2001). The protein was concentrated to 7.5 mg/ml and crystallized by the hanging drop technique against a reservoir containing 100 mM ammonium sulfate, 100 mM sodium citrate pH 5.2 and 10 mM dithiothreitol at 20°C. A selenomethionine (SeMet) derivative of the complex crystallized under similar conditions. X‐ray diffraction data (Table I) were collected at cryogenic temperatures in cryobuffer containing 35–40% glycerol, and processed with Denzo‐Scalepack (Otwinowski, 1993). Although two related monoclinic crystal forms were identified under native conditions (Table I), we were unable to identify SeMet derivatives of the strongly diffracting crystal form I, and the initial solution to the phase problem was found using crystal form II. A subset of the 56 Se atoms in the asymmetric unit (AU) was identified with the SnB program (Weeks and Miller, 1999) from anomalous differences in two independently collected peak datasets. Phase refinement with MLPHARE (Collaborative Computational Project, 1994) was carried out against anomalous differences of the peak datasets, and their isomorphous differences with a low‐resolution native dataset in crystal form II. After solvent flipping in CNS (Brünger et al., 1998), the maps displayed a clear solvent boundary and clear protein features. The model of apo‐Mad2 (Luo et al., 2000) was tentatively fitted in the electron density, and this allowed the identification of subsets of heavy atom clusters belonging to the same Mad1–Mad2 complex. This eventually led to the identification of the non‐crystallographic symmetry (NCS) operators relating the eight Mad2 monomers in the AU. The resulting density‐modified 3.3 Å maps were of excellent quality and allowed the unequivocal fitting of a Mad2 monomer. The model was used to find the position of the Mad2 monomers in crystal form I by molecular replacement with AMoRe (Collaborative Computational Project, 1994). Only three of the four Mad2 monomers could be found. The Mad1 molecules, and the fourth Mad2 monomer (which has a significantly higher average B‐factor than the other Mad2 models) were identified using program ARP/wARP in Molrep mode (Perrakis et al., 2001). Model building was carried out with program O (Jones et al., 1991). Several rounds of refinement with CNS and manual rebuilding were required to generate a final model containing residues 10–203 of the four Mad2 monomers, residues 493–579 of Mad1A and Mad1B in the first tetramer, and residues 487–579 and 485–584 of Mad1A and Mad1B in the second tetramer (Table I). 90.6% and 9.4% of residues reside within the most favoured and additional allowed regions of the Ramachandran plot, respectively, with no residues in disallowed regions.
Determination of Mad2 binding parameters
Calorimetric measurements were carried with protein samples purified as described (Sironi et al., 2001) and synthetic peptides corresponding to human Cdc20111–138 and Mad1523–550 at 25°C using a VP‐ITC titration calorimeter (MicroCal Inc.). Each titration experiment consisted of a first injection of 2 μl followed by twenty‐nine injections of 8 μl. Binding isotherms were corrected for dilution heats by subtraction of blank titrations and evaluated using the Origin software package provided with the instrument.
In vitro binding and competition assays
For the experiments shown in Figures 1 and 3, bacterial lysates containing His‐Mad2, or His‐Mad2 co‐expressed with Mad1 and its mutants were incubated with Ni‐NTA–agarose (Qiagen). Bound proteins were collected by centrifugation, washed and analysed by SDS–PAGE. The protein levels observed after IMAC purification reflect the fact that His‐Mad2 is expressed at higher levels than Mad1 from the polycistronic vector (Sironi et al., 2001). For the assays in Figure 5A, a synthetic peptide encompassing residues 111–138 of human Cdc20 was added at concentrations of 0, 6, 30 and 120 μM (lanes 8–11, respectively), to solid‐phase‐bound GST–Mad1523–550 (2 μM; lane 1) that had been previously incubated with 6 μM Mad2 (lane 2) for 1 h at 4°C in the absence of the Cdc20 peptide. The experiments with no pre‐incubation were carried out using identical concentrations of the reagents as in the pre‐incubation experiments, but the competing Cdc20 peptide (lanes 3–6; same concentrations as in lanes 8–11) was added to solid‐phase‐bound GST–Mad1523–550 before addition of Mad2. After mixing the three reagents, both reactions were protracted for the indicated time at room temperature (RT). The beads were centrifuged, washed, and bound proteins analysed by SDS–PAGE. For the experiments of Figure 5B–D, the short Mad1–Mad2 core complex, Mad2R133A (Sironi et al., 2001), and a Cdc20111–150 synthetic peptide were analysed on a Superdex200 column on a SMART system (Amersham‐Pharmacia). To evaluate peptide binding to the short Mad1–Mad2 core complex or Mad2R133A, these species were mixed and incubated at RT for different times, and finally separated on the same column.
Accession numbers
Coordinates of Mad1–Mad2 are available from the Protein Databank (ID code 1GO4).
Thanks to Kevin Hardwick, Kristian Helin, Jan‐Michael Peters, Simonetta Piatti and members of the Musacchio group for critical reading of the manuscript and discussions, and to Hongtao Yu for sharing coordinates of Mad2–MBP1. Also thanks to Andy Thompson and Ed Mitchell (European Synchroton Radiation Facility) and Massimo Degano and Alberto Cassetta (Sincrotone Trieste) for support in data collection. A.M. is an EMBO Young Investigator and a Scholar of the Italian Foundation for Cancer Research. This work was supported by a grant from the Italian Association for Cancer Research to A.M. We also wish to acknowledge the generous support of the Giovanni Armenise–Harvard Foundation for Advanced Scientific Research.
- Copyright © 2002 European Molecular Biology Organization


text:
Optical reconstruction of transparent objects with phase-only SLMs
Stoykova, E.
Yaraş F.
Yontem, A.Ö.
Kang H.
Onural L.
Hamel P.
Delacrétaz, Y.
Bergoënd I.
Arfire, C.
Depeursinge, C.
Date Issued2013
Journal Title
Optics Express
Optical Society of American (OSA)
Research Paper
Please cite this item using this persistent URLhttp://hdl.handle.net/11693/20716
Three approaches for visualization of transparent micro-objects from holographic data using phase-only SLMs are described. The objects are silicon micro-lenses captured in the near infrared by means of digital holographic microscopy and a simulated weakly refracting 3D object with size in the micrometer range. In the first method, profilometric/tomographic data are retrieved from captured holograms and converted into a 3D point cloud which allows for computer generation of multi-view phase holograms using Rayleigh-Sommerfeld formulation. In the second method, the microlens is computationally placed in front of a textured object to simulate the image of the textured data as seen through the lens. In the third method, direct optical reconstruction of the micrometer object through a digital lens by modifying the phase with the Gerchberg-Saxton algorithm is achieved. © 2013 Optical Society of America.
Published ashttp://dx.doi.org/10.1364/OE.21.028246
- Research Paper [7144]


text:
Monthly political analysis on nations in
economic or political transition
Undoubtedly one of the most important geopolitical events of this year, perhaps this decade, has been the deserved re-election of the US president.
Newnations offers its 'take' on this with particular emphasis on Foreign Policy, which we believe will reflect the views of many concerned non-US citizens. At the time of President Obama's first election victory, the relief to see the end of the disastrous eight years of Bush 43, was tangible throughout much of the world –including in some of those nations not usually to be found in the American camp.
There is little question, based on the well-reported Republican primaries, that Mitt Romney characterised by such comments as: 'Russia was the US's main geopolitical adversary', indicated that another foreign affairs 'know-nothing' might next occupy the White House. The result has meant that once again, the presidency is to be at least pragmatic not ideological, and once again a great relief is the uppermost early response.
We would hope that the following report by Newnations Associate editor, Peter Crisell, might in it's main content, resemble the State Department position paper that President Obama will be reading following his return to office. Whatever else, there is much to do and a second term is perhaps the time to do it!